Oxidative Dearomatization of PLP in Thiamin Pyrimidine Biosynthesis in Candida albicans

The yeast thiamin pyrimidine synthase THI5p catalyzes one of the most complex organic rearrangements found in primary metabolism. In this reaction, the active site His66 and PLP are converted to thiamin pyrimidine in the presence of Fe(II) and oxygen. The enzyme is a single-turnover enzyme. Here, we report the identification of an oxidatively dearomatized PLP intermediate. We utilize oxygen labeling studies, chemical-rescue-based partial reconstitution experiments, and chemical model studies to support this identification. In addition, we also identify and characterize three shunt products derived from the oxidatively dearomatized PLP.


Overexpression and Purification of THI5p and its mutants
E.coli BL21(DE3) cells containing the THI5p gene in the pET28b vector were grown in minimal media (11.3 g M9 salts, 13.3 mL 50% glucose, 2.7 mL 1 M MgSO4, 100 µL 1 M CaCl2 diluted to 1 L) containing kanamycin (40 mg/mL) with shaking at 37 °C until the OD600 reached 0.6 (without supplementation of any externally added Fe-salt to minimize the production of inactive protein during overexpression and purification process).Protein overexpression was then induced with IPTG (final concentration of 500 µM), and cell growth was continued at 15 °C for 16 h.The cells were harvested by centrifugation, and the cell pellets from 1 L of culture were resuspended in 20 mL of lysis buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, 5 mM DTT, pH 8.0) and lysed by sonication (Heat System Ultrasonics Model W-385 sonicator, 1.5 s cycle, 60% duty).The resulting cell lysate was clarified by centrifugation and the THI5p protein was purified on a Ni-NTA column following the manufacturer's instructions.After elution, the protein was desalted under anaerobic conditions using an Econo-Pac 10DG column (BioRad) preequilibrated with 100 mM Tris-HCl buffer, 5 mM DTT, 30% glycerol, pH 7.5.The purified protein was stored in aliquots in liquid nitrogen.All mutants used in this study (H66G and K62A) were also purified using this protocol.

N-THI5p overexpression and purification
The overexpression and purification procedures were the same as described above for THI5p except 14 NH4Cl was replaced with 15 NH4Cl in the M9 salts.

C-15 N-THI5p overexpression and purification
The overexpression and purification process for 13 C-15 N-THI5p was the same as described above except glucose and ammonium chloride were replaced with 13 C-glucose and 15 N-ammonium chloride.

Overexpression and purification of Pyridoxal Kinase (PdxK)
E.coli BL21(DE3) cells containing the PdxK gene in the pET18b vector were grown in LB media containing ampicilin (100 mg/mL) with shaking at 37 °C until the OD600 reached 0.6.Protein overexpression was then induced with IPTG (final concentration of 500 µM), and cell growth was continued at 15 °C for 16 h.The cells were harvested by centrifugation, and the cell pellets from 1 L of culture were resuspended in 20 mL of lysis buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, 5 mM DTT, pH 8.0) and lysed by sonication (Heat System Ultrasonics Model W-385 sonicator, 1.5 s cycle, 60% duty).The resulting cell lysate was clarified by centrifugation and the PdxK protein was purified on a Ni-NTA column following the manufacturer's instructions.After elution, the protein was desalted using an Econo-Pac 10DG column (BioRad) pre-equilibrated with 100 mM Tris-HCl buffer, 5 mM DTT, 30% glycerol, pH 7.5.The purified protein was stored in aliquots in liquid nitrogen.

Reconstitution of THI5p activity
The THI5p reaction was performed in 100 mM HEPES buffer with 1mM TCEP, pH 7.5 containing THI5p(240 µM), Fe (NH4)2(SO4)2 (120 µM) and PLP (480 µM).The enzyme was anaerobically preincubated with the iron for 30 mins in an ice bath.Then PLP was added followed by another anaerobic incubation at room temperature for 30 mins.Finally the mixture was aerobically incubated at room temperature for 3h.The reaction was quenched by filtering with a 10 kDa cut-off filter.

THI5p reaction with 18 O2
THI5p reaction was carried out as described above except during the aerobic incubation part the reaction mixture was exposed to 18 O2 rather than 16 O2.

Trapping of the PLP-derived by-product with phenylhydrazine
After the THI5p reaction is quenched by ultrafiltration, 10 μL of phenylhydrazine stock solution (5 μL in 1 mL of water, 50 mM) was added to the filtrate.The mixture was incubated at 37 °C for 2.5 h followed by LC-MS analysis (LC-MS condition 1). 2,3

Synthesis of 27
The synthesis of 27 was carried out as previously reported. 2,3 ure S2.Predicted oxygen labelling pattern in PLP by-product 27 (general structure without any labelling is shown in the inset).The oxygen labelling pattern is shown in two ways considering the possibility of solvent exchange.a) and b) Oxygen labelling pattern when the reaction was run in 50% H2O 18 .c) and d) Oxygen labelling pattern when 18 O2 is used.e) and f) Oxygen labelling pattern with 18 O2 and [4',5'-  16  16 Predicted Oxygen Labelling Pattern in 18

Synthesis of 31
To a solution of starting pyridoxal phosphate (10 mmol) in DMSO (25 mL) was added SIBX (11 mmol) as a solid in one portion.The resulting suspension was stirred at room temperature for 24 hours, after which time TFA (780 µL) was added, and the mixture was further stirred for 12 hours.The reaction mixture was then diluted with water (50 mL) and neutralized with a saturated solution of aq.Na2CO3. 4The reaction mixture was CIP treated before HPLC analysis and purification (HPLC condition 5) and LC-MS (condition 1) analysis.Mass in negative mode 365.0989Da.

Enzymatic reaction condition for P7.8 formation
The reaction conditions for P7.8 formation were the same as those used for the THI5p reconstitution reaction described above.HPLC conditions 3 and 4 were used for the analysis of P7.8 formation, isolation, and purification.LC-MS analysis (LC-MS condition 1) was carried out after dephosphorylation by CIP treatment of the reaction mixture.

P7.8 formation from isotopically labeled THI5p
The reaction conditions for P7.8 formation were the same as those used for the THI5p reconstitution reaction described above except for the replacement of THI5p with 13 C-15 N-or 15 Nlabelled protein.LC-MS analysis (condition 1) of the reactions showed a 1 Da increase in the mass of P7.8 (from 165.0664 Da to 166.0629 Da).

Quantitation of Cyano-PLP (33) formed in the THI5p reaction
For the quantitation of cyano-PLP a calibration curve was made by plotting peak area vs. concentration (HPLC condition 1) of synthesized cyano-pyridoxal.Then the peak area of the cyano-pyridoxal (~800 AU) from the THI5p reaction (obtained after dephosphorylation/CIP treatment) was fit to the equation derived from the calibration curve.In this way, it was determined  Co-migration that 65 µM of cyano-PLP is formed which is ~ 27% of the enzyme concentration (240 µM THI5p was used) in the reaction (protein concentration was determined by absorbance measurement, the extinction coefficient of the protein was calculated as 50,310 M -1 cm -1 using Protparam tool from Expasy (https://web.expasy.org/protparam/).

Conditions for trypsin digestion of single-turnover inactivated THI5p
The THI5p reaction mixture was desalted using a Bio-Spin 6 column (Bio-Rad) preequilibrated with 25 mM ammonium bicarbonate, pH 8.0.The inactive enzyme (50 µg, 1.8 µL of 660 µM THI5p) was added to 10 µL of 6M guanidine-HCl, 25 mM ammonium bicarbonate, pH 8.0.DTT (1 µL of 200 mM solution in 25 mM ammonium bicarbonate, pH 8.0) was added and the resulting mixture was incubated at room temperature for 1 hour.After incubation 10 µL of 200 mM iodoacetamide in 25 mM ammonium bicarbonate, pH 8.0, was added and the reaction mixture was incubated at room temperature in the dark for 1 hour.Finally, 77.5 µL of 25 mM ammonium bicarbonate, pH 8.0 was added to dilute the guanidine-HCl to 0.6M followed by addition of 1 µg of trypsin.[8]

Conditions for trypsin digestion of methoxyamine-derivatized THI5p (38)
After the completion of the THI5p reaction (carried out in presence of catalase, THI5p: catalase = 1:1000), excess methoxyamine hydrochloride (2 µL of 1.2 M solution) was added and the reaction mixture was incubated at 37 o C for 2 hours and desalted using a Bio-Spin 6 column (Bio-Rad) pre-equilibrated with 25 mM ammonium bicarbonate, pH 8.0.The inactive enzyme (50 µg, 1.8 µL of 660 µM THI5p) was added to 10 µL of 6M guanidine-HCl, 25 mM ammonium bicarbonate, pH 8.0.After DTT addition (1 µL of 200 mM DTT in 25 mM ammonium bicarbonate, pH 8.0) the resulting mixture was incubated at room temperature for 1 hour.Iodoacetamide (10 µL of 200 mM solution in 25 mM ammonium bicarbonate, pH 8.0) was then added and the reaction mixture was incubated at room temperature in the dark for 1 hour.Finally, 77.5 µL of 25 mM ammonium bicarbonate, pH 8.0 was added to dilute the guanidine-HCl to 0.6M followed by

Enzymatic reaction condition for chemical rescue experiment with THI5p-H66G
The chemical rescue experiment was performed with the THI5p-H66G mutant.][11][12][13] The enzyme was anaerobically preincubated with Fe(NH4)2(SO4)2 in an ice bath for 30 min.Then PLP was added, and the reaction mixture was incubated for another 30 minutes at room temperature.Imidazole was then added, and the reaction was aerobically incubated at room temperature for 4 hr.The reaction was quenched by filtering with a 10 kDa cut-off filter and analyzed by HPLC (condition 1) and LC-MS (condition 1, LC-MS was done after dephosphorylation by CIP treatment of the sample).

Dephosphorylation of the THI5p-H66G reaction product
After completion of the THI5p-H66G-catalyzed reaction, the reaction mixture was treated with CIP (20 units) and incubated at 37 o C for 2 hr.Then the mixture was quenched by filtering with a 10 kDa cut-off filter and analyzed by HPLC (condition 1) and LC-MS (condition 1).The chemical rescue reaction was carried out as described above except 16 O2 was replaced with 18 O2.

2-D-imidazole labeling of P9.2
The chemical rescue reaction was carried out as described above except imidazole was replaced with 2-D-imidazole. 14

Synthesis of deuterated Pyridoxal Phosphate (mixture of 2'-CD3-PLP and 4'formyl-D-2'-CD3-PLP)
Deuterated pyridoxal was synthesized, with some minor modifications, as previously reported (Figure S22). 1,15 or the enzymatic phosphorylation, 10 µL of 10 mM pyridoxal was added to 5 µL 60 µM pyridoxal kinase (PdxK) in 53 µL of 100 mM HEPES buffer (pH 7.5).Then 20 µL of 10 mM ATP and 2 µL of 0.1M MgSO4 were added.The reaction mixture was incubated at 37 o C for 5 hours and then filter quenched with 10 kDa cut-off filter.Labeled PLP was purified by HPLC (Condition 2) as a mixture of 72a and 72b.After HPLC purification, for LC-MS analysis (condition 1) they were dephosphorylated to improve the flying efficiency.

Synthesis of 52
8][19] After following the synthetic scheme and HPLC purification (condition 3), it was used as a reference for the identification of the dephosphorylated THI5p-K62A reaction product (P16.9)(HPLC condition 3 and LC-MS (condition 1).Mass in positive mode 199.0724.Characterization of P15.9 from the THI5p-K62A catalyzed reaction Pyridoxal nitrile oxide from the THI5p-K62A reaction was dephosphorylated and purified by HPLC (condition 4).It was derivatized by reaction with pyridoxal (61) in water to form the 1,3dipolar reaction product (78).The reaction mixture was then heated at reflux in the presence of t-BuOK for 2 hours followed by neutralization and solvent removal. 20The resulting reaction mixture containing cyano pyridoxal (34) and pyridoxoic acid (62) was analyzed by HPLC (condition 6) and LC-MS (condition 1) and used for the co-migration studies.

Synthesis of 2'-CD3-[48]
2'-CD3-[48] was prepared following Scheme (a) in Figure S37. 15,16 or the enzymatic phosphorylation, 10 µL of 10 mM pyridoxal was added to 5 µL 60 µM pyridoxal kinase in 53 µL of 100 mM HEPES buffer (pH=7.5).Then 20 µL of 10 mM ATP and 2 µL of 0.1M MgSO4 were added.The reaction was incubated at 37 o C for 5 hour and then filter quenched with a 10 kDa cutoff filter.The filtrate was treated with hydroxylamine as described above for the preparation of 48 and the resulting oxime was HPLC purified (condition 4) and analyzed by LC-MS (condition 1) after CIP treatment.Mass in positive mode after dephosphorylation is 186.0966Da.

Figure
Figure S6.LC-MS analysis of 31.a) Mass of purified 31.b) MS/MS fragmentation spectra of 31 showing the mass of its monomer 60 as the predominant fragment peak.

Figure S9 .Figure S10 .
Figure S9.Identification of the shunt metabolite in the native THI5p reaction.a) HPLC chromatogram showing the formation of HMP-P and the P7.8 metabolite in the native THI5p catalyzed reaction.b) Comparison of the UV-Vis spectra of CIP treated P7.8 with pyridoxal, pyridoxal analogs and HMP.c) Structures of all compounds used for the UV-Vis comparison.

Figure
Figure S11.LC-MS analysis of P7.8 formation with isotopically labeled THI5p.a) EIC of mass 166.0629 in 13 C-15 N THI5p full reaction along with controls.b) EIC of mass 166.0629 in the 15 N THI5p full reaction along with controls.c) and d) Mass spectra of P7.8 (after dephosphorylation) obtained in 13 C-15 N THI5p full reaction and 15 N-THI5p full reactions respectively.

Figure S12 .Figure S13. 1 H
Figure S12.Synthesis of 34.a) Synthetic scheme for the preparation of 34.b) Mass spectrum of purified 34.

Figure S16 .
Figure S16.Quantitation of Cyano PLP in native THI5p reaction.a) HPLC chromatogram of synthesized cyano-pyridoxal standard in different concentrations ranging from 1 mM to 0.03 mM B) Calibration curve obtained by plotting peak area vs. concentration of Cyano-pyridoxal for the quantification of Cyano-PLP.

Figureb 1 V
Figure S17.MS/MS analysis of 38.a) Mass increment for the conversion of the Lys62-containing peptide to 38 is 27.9949 Da. b) Calculation of the mass increment.c) MS-MS fragmentation of the modified peptide 38.This fragmentation pattern is consistent with the predicted aldehyde modification on Lys62.The fragmented peptide sequence for b and y ions are also shown.Modified Lys 62 residue is indicated in red as K*.

Figure S18 .
Figure S18.HPLC analysis of THI5p-H66G reaction.a) Imidazole concentration dependence of the formation of P9.2.b) UV spectrum of P9.2.c) HPLC chromatogram for the anaerobic control (reaction run in absence of oxygen) of the THI5p-H66G reaction showing that formation of P9.2 requires oxygen.

Figure S19 .
Figure S19.Dephosphorylation and LC-MS analysis of the THI5p-H66G reaction.a) Dephosphorylation of the THI5p-H66G reaction product P9.2 to give P23.b) Extracted Ion Chromatograms (EIC) of the mass 250.0833 in THI5p-H66G reaction (after dephosphorylation) showing the formation of dephosphorylated P9.2 in the full reaction only.
-imidazole was synthesized according to the procedure reported by Proniewicz et al.14

Figure S23 .
Figure S23.HPLC chromatogram at 360 nm of the pyridoxal (71a and 71b) phosphorylation reaction mixture indicating the formation of 72a and 72b.

FigureFigure S25 .Figure S26. 1 H
Figure S24.LC-MS analysis of the mixture of 72a and 72b after HPLC purification and dephosphorylation (by CIP).a) Extracted ion chromatograms of deuterated pyridoxal phosphates (72a and 72b) after HPLC purification and dephosphorylation in the form of 71a and 71b.b) Mass spectrum of pyridoxal phosphates after HPLC purification and dephosphorylation.

Figure S30 .Figure S31. 1 H
Figure S30.Synthesis of 52.a) Synthetic scheme for the preparation of 52.b) Mass of synthetic standard of 52.c) HPLC chromatogram at 360 nm showing co-migration of dephosphorylated P16.9 with 52.

Figure S33 .FigureFigure
Figure S33.Characterization of P15.9.a) Strategy for the characterization of P15.9 by its conversion to 34. b) and c) Mass spectra of the products obtained following scheme (a).

Figure S36 .
Figure S36.MS/MS fragmentation spectra of dephosphorylated and HPLC purified P15.9 in negative mode showing additional support for its structural assignment.

Figure S37 .
Figure S37.Synthesis of 2'-CD3-[48].a) Scheme for synthesis of 2'-CD3-[48].b) HPLC chromatogram at 360 nm showing the enzymatic phosphorylation forming 72a in the full reaction mixture compared to the indicated controls.c) HPLC chromatogram at 360 nm of the phosphorylation reaction mixture treated with hydroxylamine to form 2'-CD3-[48] in full reaction compared to the indicated controls.